The Major Histocompatibility Complex (MHC) class I pathway processes foreign cytosolic proteins into antigenic peptides, which are subsequently displayed on the surface of an infected cell and recognized by protective Cytotoxic T Lymphocytes (CTLS). The intracellular bacterial pathogen, Listeria monocytogenes, secretes the antigenic p60 protein into the host cell cytosol, where it is very efficiently degraded to immunogenic epitopes via the class I pathway. Despite detailed quantitative analyses of p60 degradation rates and processing efficiencies, little is known about the precise location of p60 in the cell, both before and during its proteolytic degradation. This proposal aims to determine the intracellular location of p6O, employing several different approaches. p60-specific antibodies and immunofluorescent methodologies will be utilized, and a variety of cell fixation and staining procedures will be tested. Another approach will be to examine the location of p6O inside living cells, through the construction of p60-Green Fluorescent Protein fusions. This technique will allow both the spatial and temporal observation of an antigenic protein targeted for degradation. In addition, potent inhibitors of proteasomal activity will be applied to determine if there exist specialized degradation sites. These investigations may provide insight into the subcellular localization of secreted bacterial proteins, may contribute to our understanding of events in the MHC class I antigen processing pathway, and may provide important information for the design of immunological therapies.